The astroglial excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) is one of the most important functional synaptic proteins in astroglia and plays important physiological and pathological functions in CNS. The regulation of EAAT2/GLT1 expression has become an excellent model for understanding how neuronal signals coordinate astroglial function at synapses. Exosomes are a class of newly identified membrane vesicles (40-100nm) of endosomal origin that are secreted from cells. They contain various biomolecules, including proteins, lipids, mRNAs and microRNAs (miRNAs). Exosome-mediated communication and its physiological significance in the CNS are essentially unknown. Based on our recently published results, we hypothesize that exosome-mediated transfer of mir-124a from neurons to astroglia increases GLT1 protein expression by suppressing astroglial miRNAs that inhibit GLT1 mRNA 3' UTR function and reduce GLT1 protein expression in astroglia. In this application, we will focus on the following aims: 1) Identify astroglial miRNAs involved in exosome mir-124a-dependent GLT1 expression regulation in astroglia we will identify astroglial miRNAs that are significantly down-regulated by neuronal exosome treatment and miR-124a transfection using miRNA microarrays. We will then validate expression changes of identified astroglial miRNAs using QRT-PCR and determine whether these astroglial miRNAs directly inhibit GLT1 3' UTR function using a luciferase based assay. Finally, we will determine whether these identified astroglial miRNAs reduce GLT1 protein expression levels and GLT1-dependent glutamate uptake in astroglia. 2) Investigate the exosome and mir-124a transfer from neurons to astroglia in vivo we will investigate the in vivo exosome and mir-124 transfer from neurons to astroglia using genetic and labeling approaches. We will generate transgenic mice that selectively express eGFP-tagged CD63 (green fluorescence) in neurons to characterize the temporal and spatial profile of in vivo transfer of neuronal exosomes (indicated by the eGFP-labeled CD63) to astroglia in intact CNS. We will also selectively deliver Alexa 750 (far red fluorescence) labeled mir-124a to motor neurons by femoral nerve injection and retrograde transport, and then examine its transfer from motor neurons to astroglia in vivo. In summary, this project will identify specific miRNAs that modulate GLT1 protein expression. These newly identified miRNAs will provide novel approaches for modulating synaptic function and are promising targets for developing GLT1-based therapeutics for the treatment of neurological diseases/injuries. In addition, this study will characterize a novel exosome-mediated signaling pathway from neurons to astroglia that regulates astroglial GLT1 expression. This intercellular pathway provides new knowledge about how neuronal signals coordinate astroglial functions at synapses.